Huda Akil - US grants

The present proposal continues our efforts aimed at understanding the expression, regulation and function of the Beta-Endorphin/ACTH precursor, proopiomelanocortin (POMC) in pituitary and in brain. This precursor gives rise to several biologically important substances including the stress hormone ACTH and the potent opioid analgesic, Beta-Endorphin (BetaE). In the past several years, we have examined the effects of various challenges, particularly stress, on pituitary POMC and have achieved some understanding of the cellular control and regulation of this precursor. We have described the interplay between pre-translational and post-translational mechanisms in maintaining the homeostasis of POMC cells in the anterior and intermediate lobes of that gland. As these mechanisms exhibit different time dependencies, they can lead to the production of unique combinations of peptide end-products as a function of the regulatory history of the POMC cell. This endows the system with a previously unforeseen level of plasticity. We have also begun to examine the pituitary POMC cells in the context of the overall circuit which controls their responsiveness to stress, the limbic-hypothalamo-pituitary adrenal axis. Finally, we have shown that the brain POMC systems shares common regulatory mechanisms with the pituitary, as revealed by mRNA changes following stress, and biosynthetic changes following chronic morphine administration. However, the neuronal regulation of these systems in the context of complex circuits and unknown inputs remains to be fully explored. This proposal builds toward a better understanding of the brain POMC systems (arcuate and NTS) by ascending from a molecular level of discourse, to a more cellular level and finally to the level of brain circuitry. At the molecular level, we shall examine the structural determinants of POMC, which lead to its highly ordered tissue-specific processing, using gene transfer and site-directed mutagenesis. At the cellular level, we shall study, in anterior pituitary cells, the consequences of activation by secretagogues and inhibition by glucocorticoids on various putative transacting factors which lead to changes in POMC transcription and levels of expression. At the integrative level, we shall describe the brain elements of the circuit which controls anterior lobe POMC, and study the coordinate regulation, at the mRNA and peptide level, of the stress- responsive hypothalamic secretagogues, CRH and vasopressin. This knowledge base will serve to guide us in our studies of brain BetaE systems at a cellular level, and in an anatomical context. We shall attempt to understand the dynamics of these systems in biosynthetic terms (mRNA levels, peptide forms, processing enzymes, releasability), starting with stress as a challenge which also engages brain POMC. We shall extend these studies to other challenges, particularly to studying the effects of analgesic electrical stimulation, and chronic administration of opiate agonists. It is hoped that this multipronged approach can yield valuable information on the role of Beta-Endorphin and related peptides in mechanisms of stress, pain and drug addiction.

The present proposal continues our efforts aimed at understanding the expression, regulation and function of the Beta-Endorphin/ACTH precursor, proopiomelanocortin (POMC) in pituitary and in brain. This precursor gives rise to several biologically important substances including the stress hormone ACTH and the potent opioid analgesic, Beta-Endorphin (BetaE). In the past several years, we have examined the effects of various challenges, particularly stress, on pituitary POMC and have achieved some understanding of the cellular control and regulation of this precursor. We have described the interplay between pre-translational and post-translational mechanisms in maintaining the homeostasis of POMC cells in the anterior and intermediate lobes of that gland. As these mechanisms exhibit different time dependencies, they can lead to the production of unique combinations of peptide end-products as a function of the regulatory history of the POMC cell. This endows the system with a previously unforeseen level of plasticity. We have also begun to examine the pituitary POMC cells in the context of the overall circuit which controls their responsiveness to stress, the limbic-hypothalamo-pituitary adrenal axis. Finally, we have shown that the brain POMC systems shares common regulatory mechanisms with the pituitary, as revealed by mRNA changes following stress, and biosynthetic changes following chronic morphine administration. However, the neuronal regulation of these systems in the context of complex circuits and unknown inputs remains to be fully explored. This proposal builds toward a better understanding of the brain POMC systems (arcuate and NTS) by ascending from a molecular level of discourse, to a more cellular level and finally to the level of brain circuitry. At the molecular level, we shall examine the structural determinants of POMC, which lead to its highly ordered tissue-specific processing, using gene transfer and site-directed mutagenesis. At the cellular level, we shall study, in anterior pituitary cells, the consequences of activation by secretagogues and inhibition by glucocorticoids on various putative transacting factors which lead to changes in POMC transcription and levels of expression. At the integrative level, we shall describe the brain elements of the circuit which controls anterior lobe POMC, and study the coordinate regulation, at the mRNA and peptide level, of the stress- responsive hypothalamic secretagogues, CRH and vasopressin. This knowledge base will serve to guide us in our studies of brain BetaE systems at a cellular level, and in an anatomical context. We shall attempt to understand the dynamics of these systems in biosynthetic terms (mRNA levels, peptide forms, processing enzymes, releasability), starting with stress as a challenge which also engages brain POMC. We shall extend these studies to other challenges, particularly to studying the effects of analgesic electrical stimulation, and chronic administration of opiate agonists. It is hoped that this multipronged approach can yield valuable information on the role of Beta-Endorphin and related peptides in mechanisms of stress, pain and drug addiction.

This application seeks continued support for the specialized training of a subset of doctoral students enrolled in the interdisciplinary Neuroscience Graduate Program at the University of Michigan. Since its inception in 1970, the Neuroscience Graduate Program has served as a focus for Neuroscience research and activities on campus. This training grant has played a key role in supporting graduate education in Neuroscience since it was first funded in 1972. It is the only training, grant on campus which exclusively supports Neuroscience Program students. The Neuroscience Program derives institutional support from several academic units on campus including the Medical School, Rackham Graduate School and the College of Literature, Science and Arts. The Neuroscience Program is comprised of 74 faculty and 30 students who participate in the administration, teaching, admissions and research training efforts of the Program. A major Neuroscience Program curricular revision is nearing completion in order to coordinate courses with the reorganization of the Medical School departments into the Program in Biomedical Sciences (PIBS). Graduate students entering the Medical School PIBS program will take the first year PIBS curriculum, perform research rotations, and at the end of their first year will choose a department or program for their Ph.D. Thus, beginning in the Fall of 1999, this training grant will draw from two pools of students, those admitted directly to the Neuroscience Program and those who enter the program after a first year in the PIBS program. The area of specialization for this NIMH Training Grant in Molecular Neurobiology will be the study of signal transduction mechanisms in the nervous system and 35 faculty from 10 departments across the University constitute the training faculty. These faculty direct vigorous, well-funded research programs and have an established history of published collaborations. Therefore, even within the field of neurobiological signal transduction, trainees are afforded a wide variety of research training experiences. Students recruited to this training grant will satisfy all the requirements of the Neuroscience Graduate Program in addition to specific requirements of this training grant. The NIMH Training Grant in Molecular Neurobiology will continue to be administered by a Director, Co-Director, and Steering Committee that function independently but in cooperation with the Neuroscience Program administration. Support for 5 trainees per year is requested. Appointment as a trainee requires not only admission to the Neuroscience Graduate Program but also sufficient interest and preparation for training in the neurobiology, of signal transduction as determined by the Steering Committee for this grant.

The purpose of this application is to investigate, at the molecular and integrative levels, the biology of the newly cloned mu and delta opioid receptors, attempting to understand how these molecules function as units, and how they interact with other brain molecules to modulate critical functions such as pain control and drug reward. A major emphasis is to understand the function of these receptors in the context of the multiplicity of ligands in the endogenous opioid family. Given that mu and delta receptors interact, to varying extents, with the products of the three opioid precursor gene family, and given that the endogenous ligands share a common "message" sequence, the question arises as to how the receptors achieve their affinity and selectivity for the various ligands. Thus, the first specific aim of this application is to carry out structure-function studies of the mu and delta receptors, investigating the nature of the binding pocket for the peptides and alkaloids, and the mechanisms of discrimination or selectivity among the various type of agonist ligands, and between agonists and antagonists. These studies will be conducted using molecular modeling and site-directed mutagenesis, as well as relying on an empirical approach with construction of chimeras to indicate critical domains, followed by mutagenesis of unique residues within these domains. The second specific aim is to place these opioid receptors in their anatomical context and to describe their expression relative to the expression of the 3 opioid precursors. Using dual in situ hybridization, receptor immunohistochemistry, or a combination of in situ and immunohistochemistry, we will study the expression of the mu and delta receptors and their ligands in order to determine the existence of autoreceptors, and to describe the interface between the opioid transmitters and the two receptors. We will survey the brain and spinal cord, and then focus on the anatomy of the opioid system in areas implicated in nociception and analgesia. This information will form the foundation of regulatory studies whereby stimuli known to alter pain responsiveness will be used to modulate the expression of the opioids, the receptors, or both. The third specific aim will move our knowledge of the mu and delta receptors at the molecular and anatomical level to a more complete description of the anatomy of drug reward, the role of opioid receptors in the reward circuits, and the modulation of these molecules by treatment with drugs such as morphine and cocaine. Using a combination of anatomical tools, we shall describe the co-expression of opioid molecules with other molecules relevant to reward mechanisms, particularly dopamine receptors. We shall define, at a finer level, subpopulations of cells in the nucleus accumbens with unique combinations of these molecules, and will study their projections. Then we shall relate these anatomical/biochemical entities to drug responsiveness, and determine changes in expression under conditions which promote drug tolerance versus drug sensitization. Taken together, these studies will permit us to use the cloning of the opioid receptors to achieve a better understanding of the biology of opioids and their role in drug use and abuse.

This proposal attempts to address the question: is the dysregulation of the Limbic-Hypothalamo-Pituitary-Adrenal (LHPA) Axis observed in depression a result of the stress which either precipitated the depression and/or resulted from it, or does the LHPA axis play a more fundamental role in the biology of depression? If the latter is correct, we would hypothesize that individuals who are prone to depression may respond to psychological stressors differently as monitored by endocrine correlates, and that they may do so not only when in episode, but also when out of episode. It would suggest that information which is laden with negative affect may be remembered differently by depressive individuals, as compared to normal controls. We propose to test these ideas by subjecting depressed patients, in and out of episode, to a social stressor and testing how they respond to it endocrinologically, how rapidly they terminate this response, and how well they habituate to its repeated presentation. We shall also use a newly devised memory test which contrast the ability to remember neutral vs. negative material, and ask whether depressed subjects, be they in or out of episode, show differences from controls in the way they handle emotional material. In addition, we shall test subjects out of episode at the purely neuroendocrine level using some sensitive challenges which we have devised and validated, and which we believe are reflective of the neuronal rather than the peripheral elements of the LHPA axis--these will include a metyrapone test in the evening at the nadir of the rhythm where we observe substantial dysregulation in drive level, and a test of fast feedback in the morning, at the peak of the rhythm, where we have shown a disturbance in glucocorticoid feedback. A complementary approach to addressing this central question is to study a group of subjects undergoing a very stressful life situation ( genetic test for a familial Breast Cancer Gene-BRCA1, to determine whether or not they carry a mutation which gives them an almost 90% chance of developing breast and/or ovarian cancer); evaluating their neuroendocrine profile at that point; and determining whether it resembles that of individuals with major depression. We shall then ask whether an abnormal neuroendocrine profile is correlated with a personal or a family history of major depression, or with the subsequent occurrence of depression or mood disorders, as determined with a 6 month follow-up. Together, this series of studies should shed light on the extent of the relationship between major depression and the dysregulation of stress responsiveness.

The focus of this proposal is to study how the limbic-hypothalamo- pituitary-adrenal (LHPA) axis changes as a function of developmental events, genetic predisposition and exposure to repeated stress, and how these factors contribute to individual differences in stress responsiveness throughout life, including during the aging process. Exactly when and how the stress exposure takes place may lead to different consequences on the endocrine, molecular, neuronal and behavioral functioning of the animal. Understanding the "plasticity" of the LHPA axis is important in helping us describe how the system maintains optimal responsiveness to stress, and how it incorporates past experience into the coping process. Thus we shall explore these issues via 3 interrelated sets of studies: 1) In a series of developmental studies, we shall examine the neuronal bases of the stress hyporesponsive period (SHRP) in the rat and the mechanisms associated with the animal's emergence from it into stress responsiveness. We shall look at the consequences of 4 models of early stress (maternal deprivation with various timing, and changes in litter size) on the duration of the SHRP, and on stress responsiveness in adult life, focusing on relating changes in the brain circuits to alterations in endocrine profiles and in learning, memory and response to novelty; 2) In studies focused on aging, we shall examine some of the determinants of individual differences in stress responsiveness in aged animals in order to understand why some animals are impaired and others are not as they age. We shall study the effect of genetic predisposition on the aging process by contrasting two strains of rats, Fisher and Lewis, which are known to have different stress response profiles. We shall also study how events taking place during development may impact on the aging process, by studying the consequences of the developmental models as the animals age, including effects on stress responsiveness, brain responsiveness and learning and memory; 3) In studies focused on young adult animals, we shall investigate the neuronal mechanisms of sensitization and habituation to repeated stressors. In particular, we shall focus on the role of specific brain structures (e.g., the suprachiasmatic nucleus, and the prefrontal cortex) in contributing to this type of plasticity. Finally, we shall begin to investigate the potential role of NMDA receptors in these plastic processes. This combination of studies should provide us with a better understanding of how the stress axis is modified throughout the life of the animal, and how this contributes to individual differences in stress responsiveness and coping.

This proposal is focused on studying the newly discovered Orphanin/Nociceptin system (OFQ system), a new peptidergic system, evolutionarily related to the endogenous opioids but exhibiting distinct biochemical, anatomical and functional properties. The working hypothesis is that the OFQ systems plays a role in the regulation of the biology of stress responsiveness, the biology of reward mechanisms, and the interactions between stress and reward mechanisms as they impact on drug-related behavior. This hypothesis is based on several lines of anatomical and functional evidence gathered to date. However, much remains to be learned about some of the fundamental features of this system before pursuing these functional questions. Thus, this application proposes to determine whether stress (novelty and restraint) regulates OFQ or its associated receptor, ORL1, in particular brain regions, and to ascertain the ability of OFQ in these stress-sensitive regions to modulate the limbic- hypothalamo-pituitary-adrenal (LHPA) axis) (Aim I). It explores the anatomical features of the OFQ system in the context of the mesocorticolimbic dopaminergic system, where it appears to be highly expressed, and addresses the issue of whether or not OFQ and ORL1 are present within dopaminergic neurons, and whether they are located in circuits which could modulate dopaminergic transmission (Aim II). Finally, in the context of the above circuits, it examines the way this system is modulated by chronic administration of opiates and psychostimulants under conditions which lead to sensitization to these drugs. In turn, it asks whether OFQ is able to alter the course of drug sensitization in stressful and non-stressful conditions (Aim III). A combination of molecular, anatomical, and behavioral tools will be used to address these questions. Together, these studies will not only shed light on the functioning of this novel peptidergic system, but will also provide new insights on the important interface between stress mechanisms, dopaminergic mechanisms, and substance abuse.

DESCRIPTION (provided by applicant): Individuals vary significantly in their propensity to experiment with drugs of abuse and to become addicted to them. Some may seek drugs as an aspect of sensation seeking or risk taking behavior. Others may do so as a result of social or psychological stress. The purpose of this proposal is to investigate the neurobiological basis for these individual differences in vulnerability to abusing drugs. The basic premise is that the combination of genetic, developmental and environmental factors that lead to differences in vulnerability is expressed in the brain, and needs to be understood at that level. The primary site of expression of relevant genes is likely in the context of the emotional brain circuits that regulate responsiveness to stress and to the environment, and that become dysregulated in addiction and in mood disorders. In order to investigate the neuronal basis of individual differences in drug abuse, this proposal relies on a behavioral model that distinguishes rats that are sensation or novelty-seeking from those that are not. These sensation-seeking animals learn to administer drugs, such as amphetamine, much more rapidly although the drugs are equally rewarding to them. We plan to characterize the patterns of gene expression in rats that exhibit different phenotypes of sensation-seeking behavior. We plan to focus on stress related genes and determine the possible presence of unique neuronal phenotypes associated with sensation-seeking and drug-taking behavior. We then plan to use a model of psychosocial stress, social defeat, to study the impact of such stress on the rate of acquisition of drug taking behavior and the associated patterns of gene expression in the emotional circuits of these differing animals. These studies should begin to give us a glimpse of how gene expression and brain circuits may differ in individuals with differing tendencies to abuse drugs, and how social stress can impact on these systems, further modifying them and leading to distinctive behavioral responses.

DESCRIPTION (provided by applicant): Individuals vary significantly in their propensity to experiment with drugs of abuse and to become addicted to them. Some may seek drugs as an aspect of sensation seeking or risk taking behavior. Others may do so as a result of social or psychological stress. The purpose of this proposal is to investigate the neurobiological basis for these individual differences in vulnerability to abusing drugs. The basic premise is that the combination of genetic, developmental and environmental factors that lead to differences in vulnerability is expressed in the brain, and needs to be understood at that level. The primary site of expression of relevant genes is likely in the context of the emotional brain circuits that regulate responsiveness to stress and to the environment, and that become dysregulated in addiction and in mood disorders. In order to investigate the neuronal basis of individual differences in drug abuse, this proposal relies on a behavioral model that distinguishes rats that are sensation or novelty-seeking from those that are not. These sensation-seeking animals learn to administer drugs, such as amphetamine, much more rapidly although the drugs are equally rewarding to them. We plan to characterize the patterns of gene expression in rats that exhibit different phenotypes of sensation-seeking behavior. We plan to focus on stress related genes and determine the possible presence of unique neuronal phenotypes associated with sensation-seeking and drug-taking behavior. We then plan to use a model of psychosocial stress, social defeat, to study the impact of such stress on the rate of acquisition of drug taking behavior and the associated patterns of gene expression in the emotional circuits of these differing animals. These studies should begin to give us a glimpse of how gene expression and brain circuits may differ in individuals with differing tendencies to abuse drugs, and how social stress can impact on these systems, further modifying them and leading to distinctive behavioral responses.

Forebrain Overexpression of a Stress-Related Gene: Impact on Reactivity to Cocaine This project tests the hypothesis that the level of activity of a stress-related gene in the brain, the glucocorticoid receptor (GR) represents a molecular antecedent for increased susceptibility to drugs of abuse. Since the glucocorticoid receptor is a ligand-activated transacting factor that modulates many target genes implicated in emotional behavior and drug reward, and since it is known to mediate stress responsiveness and impact on mechanisms of neural plasticity, it represents a potential key molecular target for modulation of abuse liability. The proposed studies rely on two related mouse transgenic models we have developed: a) a constitutive GR overexpressor (GRov) with increased GR expression in the forebrain only. This animal shows altered emotional reactivity and increased sensitization to cocaine. It also shows significant alterations in the expression of genes implicated in substance abuse as well as neural plasticity genes such as growth factors; b) a new inducible/tissue-specific GR overexpressor (iGRov) in which GR overexpression in the forebrain can be controlled at various stages of development. We plan to determine whether the GRov animals, compared to their wild type littermates, exhibit alterations in hippocampal neurogenesis and morphology and/or changes in expression of neuroplasticity genes basally and after exposure to chronic cocaine. The same panel of genes used in the other three projects will be tested. Using iGRov, 'we plan to ascertain whether there are critical periods in development, includingpre- weaning and adolescence, during which lifelong responsiveness to cocaine can be altered. Finally we will ask whether a single neonatal exposure to fibroblast growth factor 2 (FGF2), which is known to enhance neurogenesis, can counteract the impact of GR overexpression on responsiveness to cocaine under control or social stress conditions. We will ascertain the neural changes associated with this manipulation in the wild type and transgenic animals. These studies will provide a mechanistic examination of the antecedents of substance abuse and will test specifically whether a balance of stress-related genes and growth factor-related genes determines susceptibility to psychoactive drugs and whether there are key developmental windows that impact on it.

This Administrative Core will oversee the coordination of the four component projects of this overall application. The core will organize regular working meetings to discuss findings and future directions across the projects. The Core will also organize visits of the External Advisory Committee on alternate years. The External Advisory Committee includes: Drs. Mary-Jeanne Kreek (The Rockefeller University), Peter Kalivas (Medical University of South Carolina) and Marc Caron (Duke University). These advisors have all agreed to serve.

DESCRIPTION (provided by applicant): Both the initial susceptibility to addiction and its consequences on brain and behavior likely depend on the interaction of genes and environment, during development and at the time of drug exposure. Animal models are needed to analyze these variables and discover the neural mechanisms of addiction. This application consists of four highly integrated projects that revolve around the central hypothesis that mechanisms of neural plasticity are key determinants of all phases of addiction--the initial susceptibility to drug-taking, its maintenance and the long-term consequences that lead to craving and relapse. Project 1 uses two lines of rats selectively bred for the novelty-seeking trait (HR vs. LR) that predicts susceptibility to drug-taking. These rats will be exposed to cocaine, followed by various periods of abstinence, and tested with or without a social stressor. Cocaine's broad impact on neural remodeling will be indexed by assessing expression of a panel of neuroplasticity genes in relevant brain circuits, and measuring hippocampal neurogenesis. Selected target genes will be further characterized. Project 2 studies the neural impact of cocaine exposure in HR-bred and LR-bred rats and asks whether prior exposure to the drug occludes the ability of the individual to profit from subsequent positive experience (enriched environment). This will be indexed by dendritic morphology, neurogenesis and gene expression. Project 3 asks whether a manipulation that enhances hippocampal neurogenesis can protect against susceptibility to cocaine under control and stressful conditions, and whether this protective effect may be different in animals with different genetic backgrounds. Project 4 asks whether increased expression of a stress-related gene, the glucocorticoid receptor (GR) increases susceptibility to cocaine. It uses two related mouse transgenic models -a constitutive GR overexpressor and an inducible GR overexpressor to determine critical periods in development with the greatest impact on drug susceptibility. This work will provide a better molecular understanding of addiction-induced brain remodeling in individuals with different propensities for drug abuse. This should lead to novel, better-tailored drug targets for treating the early impact of addictive drugs or reversing their persistent effects that can trigger relapse. PROGRAM CHARACTERISTICS

Animal Core will serve all of the proposed projects in this application. It will provide continued selective breeding and behavioral characterization of two lines of rats that have been bred for 8 generations on the basis of differences in novelty-seeking. This trait is related to differential responsiveness to drug abuse and to differences in other behavioral and neural characteristics. The Core will conduct the animal husbandry, the testing needed for continued breeding of the colony, the initial behavioral characterization (e.g. locomotor tests) of the animals prior to transferring them to the individual investigators for specific studies. Moreover, the Animal Core will maintain the two mouse lines necessary for Project 4, with either constitutive or inducible GR over-expression. It will conduct the genotyping necessary for identifying the transgenic animals and wild type littermates prior to their use for the proposed specific studies.

DESCRIPTION (provided by applicant): We propose to study the neurobiological mechanisms of affective resilience to anxiety and stress, and to identify strategies for enhancing resilience specifically in highly vulnerable individuals. Our general hypothesis is that epigenetic mechanisms are critical predisposing factors that shape responsiveness to negative valence and impact vulnerability to chronic anxiety disorders. More specifically, we focus on the Fibroblast Growth Factor 2 (FGF2) which serves as a master molecular organizer that is critical during development and continues to be active in shaping affective responsiveness throughout life. This proposal tests the hypothesis that FGF2 is an endogenous resilience molecule that is not only a target of epigenetic mechanisms but is itself a modifier of these mechanisms that, in turn, fine-tune affective responsiveness. The proposed series of studies relies on two lines of rats that we have genetically selected based on their propensity to explore a mildly stressful novel environment. Our breeding strategy over 40 generations has resulted in contrasting behavioral phenotypes that capture a stable bias towards negative valence responsiveness versus positive valence responsiveness. In particular, bred Low-Responder rats (bLRs) exhibit greater basal anxiety and depression behaviors, greater fear conditioning, greater responsiveness to chronic and social defeat stress, lower levels of hippocampal FGF2 and a distinct epigenetic profile when compared to bred High Responders (bHRs) that are significantly more resilient. Thus bLRs are a model of vulnerability to negative affective disorders and a target for resilience enhancement. Aim 1 uses direct administration of FGF2 to promote resilience during early life and in adolescence. It also investigates environmental complexity (EC) as a strategy for promoting resilience during adolescence. It studies the impact of these interventions on two molecular master organizer genes in hippocampus- FGF2 itself, which reduces anxiety and the glucocorticoid receptor GR, which enhances anxiety behavior. Aim 2 characterizes the basal epigenetic profiles associated with the bHR/bLR phenotypes and the impact of resilience induction on these profiles both at the global level and in association with FGF2 and GR promoters. These studies will be extended to anatomical analyses using a range of tools including Clarity. Aim 3 addresses at a mechanistic level the functional bidirectional relationship between FGF2 and epigenetic mechanisms and their impact on resilience--- be it basal resilience (in bHRs) or induced (in bLRs). It uses virally-mediated, targeted RNA intervention strategies to knockdown either FGF2 or histone modifying enzymes in the hippocampus and determine whether they play an essential role in the induction of resilience. Together, these studies will provide a highly targeted approach to understanding and harnessing epigenetics as key factors in affect regulation.

A long-standing challenge in basic and translational neuroscience and in clinical practice is to understand the vast inter-individual differences in vulnerability to substance abuse and addiction. To address this challenge we propose to study the genetic and functional basis of novelty-seeking behavior in two lines of rats that offer a uniquely powerful model for understanding the neural mechanisms of drug seeking, addiction and relapse. We developed these lines by selecting for high and low propensity to explore a mildly stressful novel environment, respectively. After 37 generations, the bred High Responders (bHRs) and bred Low Responders (bLRs) show contrasting spectra of behaviors, which are heritable in both lines. Compared to bLRs and outbred rats, bHRs exhibit higher novelty seeking and impulsive behaviors, lower anxiety, greater propensity to sensitize to psychostimulants, and lower thresholds for drug- and cue-induced relapse, reminiscent of human ?externalizing disorders?. The bLRs are more prone to anxious and depressive behaviors, more responsive to psychosocial stress, which triggers drug-seeking behavior. Thus, the two lines exemplify two extremes of emotional reactivity that map onto human temperamental differences and underlie two paths to drug abuse?novelty seeking and reactivity to psychosocial stress. Our working hypothesis is that functional DNA variants in a limited number of genes, initially derived from outbred Sprague Dawley (SD) founders, account for the current molecular and behavioral divergence of the two lines. Our goal is to identify these causal genes through (1) mapping of quantitative trait loci (QTL) in both an F2 cohort already collected and in SD animals that represent the founders, and (2) further integration of functional genomic data. We propose to apply several sequencing-based technologies and analytical tools under the following specific aims (SAs): SA1: Conduct genome sequencing and quantitative trait loci (QTL) analyses in a bHR-bLR intercross population (n~636, males and females) using a low-cost Genotype by Sequencing method. SA2: Identify eQTLs, allele-specific expression, and transcripts/pathways associated with the traits by using RNAseq analysis of key neural structures-- the nucleus accumbens and hippocampus. As some genes may exert their primary influence during development, we will analyze both young F2 rats (age: 28 days) and adults. SA3: Perform genomewide association study of 1,000 outbred SD rats, followed by integration of all strands of data to identify putatively causal variants and provide initial validation using qPCR, in situ hybridization, and further behavioral tests. We expect to find functional alleles at multiple genes that existed in the SDs and have evolved further apart in the two lines. Many of these genes may be directly relevant to the corresponding human phenotypes or, at a minimum, provide clues to important pathways that could explain or predict the differential vulnerability to addiction and relapse in humans. Our ultimate goal is to gain a deeper mechanistic understanding of addiction, and translate this knowledge to more precise and effective treatment for patients.

Substance use disorders (SUDs) are debilitating disorders that have a major personal and societal impact. Both prevention and treatment strategies require a better understanding of neurobiological and behavioral traits that predispose for drug taking as well as risk of addiction. Our laboratory has selectively bred rats for over 60 generations in order to select for particular behavioral traits, ultimately generating a robust behavioral model of emotional reactivity and addiction liability. Bred high responder (bHR) and bred low responder (bLR) animals represent extreme ends of emotional reactivity: bHR animals represent an externalizing phenotype, with low behavioral inhibition and high levels of impulsivity and drug-seeking behavior. bLR animals represent an internalizing phenotype, are behaviorally inhibited, with a high level of anxiety- and depressive-like behaviors, but less prone to drug seeking and relapse. Importantly the reproducibility of behavioral traits across generations allows for a predictive model and the investigation of developmental mechanisms that contribute to adult behavior. A major goal of the parent award (U01) is to understand the genetic basis of temperamental tendencies that predispose for addictive behaviors. In recent years, our lab has characterized genetic variants as well as hippocampal gene expression changes that arise early in development in the bHR/bLR model and strongly discriminate between the lines. A top candidate gene in development is the Bone Morphogenetic Protein 4 (Bmp4), which has well- established roles in patterning the developing nervous system, neurogenesis, and importantly, in specification of astrocytes and oligodendrocyte number during development. It is currently unknown whether glial specification and postnatal Bmp4 expression are important for shaping temperament and behavior. In this application we propose to study the role of Bmp4 in glial subtype specification and behavioral responses within the bHR/bLR model. We hypothesize that differences in Bmp4 arise early in development and contribute to a diverging glial phenotype in which the generation of oligodendrocytes and astrocytes is modified, and that differences in cell types, in turn, contribute to differences in temperament. This hypothesis will be tested using a combination of histological, viral, and behavioral approaches. This work has the potential to reveal a novel mechanism by which the developing hippocampus is patterned in the early postnatal window, and point to a new role for glial subtypes in shaping vulnerability to addictive and affective disorders.